In a series of seven experiments, the virulent Nichols strain of Treponema pallidum was shown to attach and replicate on the surface of tissue culture cells of cottontail rabbit epithelium (Sf1Ep) growing in conventional monolayer cultures under an atmosphere of 1.5% oxygen. Five days after inoculation of 10⁶T. pallidum, the number of treponemes had increased to between 8 × 10⁶ and 2.59 × 10⁷. The viability of harvested organisms ranged from 86 to 97%. The number of T. pallidum continued to increase, generally reaching a plateau between days 9 and 12 of incubation, with increases ranging up to 100-fold and averaging 49-fold. There appeared to be a ceiling of multiplication of about 2 × 10⁸ irrespective of the inoculum, which ranged from 10⁶ to 10⁸T. pallidum. Concurrent deoxyribonucleic acid assays were performed on the cultures containing T. pallidum to obtain further evidence of replication. Significant increases in treponemal deoxyribonucleic acid were observed when the inocula ranged from 10⁶ to 10⁷, with the greatest increases, as might be expected, being in the former group. There was also excellent correlation in the amount of deoxyribonucleic acid per treponeme; the averages for the 10⁶, 2.5 × 10⁶, and 10⁷ groups were 3.46 × 10⁻¹⁴, 3.28 × 10⁻¹⁴, and 2.79 × 10⁻¹⁴ g per treponeme, respectively (3.14 ± 0.72 × 10⁻¹⁴ g per treponeme). In each experiment, organisms were harvested from the group inoculated with 10⁶T. pallidum after 7 days of incubation to test for virulence. In all instances, the organisms were virulent; erythematous, indurated, treponeme-containing lesions were produced from an average of six to seven organisms. Scanning electron microscopy revealed that during the course of replication many microcolonies of treponemes formed on the surface of the cells.